EFFECTS OF MANGIFERIN IN IMPROVING

METABOLIC SYNDROME RISK FACTORS IN MICE

FED HIGH FAT DIET

By

CHEE YEN LAU

Bachelor of Science in Medical Bioscience

Monash University

Sunway City, Malaysia

2009

Submitted to the Faculty of the

Graduate College of the

Oklahoma State University

in partial fulfillment of

the requirements for

the Degree of

MASTER OF SCIENCE

May, 2012

ii

EFFECTS OF MANGIFERIN IN IMPROVING

METABOLIC SYNDROME RISK FACTORS IN MICE

FED HIGH FAT DIET

Thesis Approved:

Dr. Edralin A. Lucas

Thesis Adviser

Dr. Brenda J. Smith

Dr. Barbara J. Stoecker

Dr. Sheryl Tucker

Dean of the Graduate College

iii

TABLE OF CONTENTS

Chapter Page

I. INTRODUCTION ......................................................................................................1

Introduction ..............................................................................................................1

Hypothesis................................................................................................................4

Objectives ................................................................................................................4

II. REVIEW OF LITERATURE

Metabolic syndrome prevalence ..............................................................................5

Pathophysiology of obesity ......................................................................................6

Insulin resistance ......................................................................................................8

Atherosclerosis .......................................................................................................10

Hypertension ..........................................................................................................11

Peroxisome proliferator activated receptor agonists……………... .......................13

PPAR, insulin sensitivity, and dyslipidemia ..........................................................14

Adverse effects of Thiazolidinediones (TZDs) ..................................................... 15

Weight gain…………………………………………………………………. 15

Edema ..................................................................................................……..16

Bone loss ........................................................................................................16

Increased risk and death from CVD...............................................................17

Composition of mango ............................................................................................18

Polyphenolic compounds ........................................................................................20

Mangiferin...............................................................................................................21

Factors affecting the contents of antioxidant compounds in mango.......................22

Anti-diabetic and hypolipidemic effects of mangiferin ..........................................24

Antioxidant property of mangiferin ........................................................................27

Anti-inflammatory effect of mangiferin .................................................................30

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Chapter Page

III. METHODOLOGY

Animal care ............................................................................................................32

Dietary treatments ..................................................................................................32

Glucose tolerance test ............................................................................................33

Necropsy and tissue processing .............................................................................33

Clinical analyses ....................................................................................................34

Plasma insulin ........................................................................................................36

Liver and fecal total lipids……………………………………………………... ..36

Liver and adipose tissue histology .........................................................................37

Statistical analyses .................................................................................................37

IV. FINDINGS

Food intake, body and tissue weights ....................................................................41

Whole body composition .......................................................................................42

Clinical chemistry ..................................................................................................43

Glucose tolerance test ............................................................................................44

Histology ................................................................................................................45

V. DISCUSSION .........................................................................................................53

VI. REFERENCES………………………………………………………………….. 60

v

LIST OF TABLES

Table Page

1. Typical nutrient composition of mango pulp ........................................................19

2. Total phenolic and individual phenolic compound in mango pulp, peel, kernel and

stem bark. ..............................................................................................................20

3. Total phenolics, total carotenoids, β-carotene, and total ascorbic acid in the pulp of

four mango cultivars .............................................................................................22

4. Nutrient composition of freeze-dried Tommy Atkins mango used in the study…...

...............................................................................................................................38

5. Composition of the experimental diets .................................................................39

6. Effects of mango, rosiglitazone,and mangiferin on food intake and body and tissue

weights of mice fed high fed diets for two months ...............................................46

7. Effects of mango, rosiglitazone,and mangiferin on body composition, bone mineral

area (BMA), content (BMC), and density (BMD) of mice fed high fat diet for two

months ..................................................................................................................47

8. Effects of mango, rosiglitazone, and mangiferin on clinical chemistry parameters,

and liver and fecal total lipid of mice fed high fat diet for two months ...............48

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LIST OF FIGURES

Figure Page

1. Mangiferin structure………………………………………………………….21

2. Effects of mango, rosiglitazone, and mangiferin on weekly body weights of mice

fed with high fed diets for two months…………………………………….. ..49

3. Effects of mango, rosiglitazone, and mangiferin on glucose tolerance

(a) after 30 days of treatment…………………………………………………50

(b) after 60 days of treatment…………………………………………………50

(c) total glucose area under the curve…………………………………………51

4. Effects of mango, rosiglitazone and mangiferin on liver and adipose tissue

histology of mice fed high fat diets for two months…………………………..52

1

CHAPTER I

INTRODUCTION

Metabolic syndrome (MetS) is a complex disease which interlinks the risk factors for

cardiovascular disease (CVD) and diabetes [1]. According to the International Diabetes

Foundation (IDF) and the American Heart Association/National Heart, Lung, and Blood Institute

(AHA/NHLBI) [2], there are five characteristics for the clinical diagnosis of metabolic

syndrome: 1) waist circumference >102 cm and 88 cm, for men and women, respectively; 2)

triglycerides >150 mg/dL (1.7mmol/L); 3) high density lipoprotein-cholesterol (HDL-C) <

40mg/dL (1.0mmol/L) and 50mg/dL (1.3mmol/L) for men and women, respectively; 4) systolic

or diastolic blood pressure >130 mmHg or 85 mmHg, respectively; and 5) glucose >100mg/dL.

The presence of any three out of the five risk factors is considered diagnosis of MS [2]. Metabolic

syndrome is strongly associated with prothrombotic and proinflammatory states [1].

According to the Centers for Disease Control and Prevention (CDC), the prevalence of

obesity in the United States for the past 25 years (1985-2010) has increased dramatically, with

obesity rate of 25% or more in 36 states [3]. Diabetes, on the other hand, affects 25.8 million

people (approximately 8.3% of the American population) in the year 2010 [4]. An estimated of

81 million American adults were affected with one or more types of CVD in the year 2010. If this

trend continues, the health status of the US population is in jeopardy. Therefore, determination of

the underlying causes and treatments for factors associated with MetS are of utmost importance.

2

Some of the factors involved in the development of MetS are modifiable and some are not. Age

and gene inheritance are uncontrollable risk factors. However, dietary fat intake and physical

inactivity are modifiable MetS risk factors. Long-term consumption of high fat diet and sedentary

lifestyle are strongly associated with excessive accumulation of abdominal fat, which is

associated with elevated levels of circulating free fatty acids (FFAs) due to the expansion of

adipose tissues [5-7]. Obesity is accompanied with enlarged adipose tissues, leading to the

activation of certain protein kinases and inflammatory mediators which inhibit the insulin actions.

Impaired insulin actions will lead to elevated levels of FFAs and over time, increased

concentration of circulating FFAs lead to the accumulation of lipids or plaque formation in the

blood vessel and hence formation of CVD. Hypertension is another MetS risk factor and a

contributor to the development of CVD. Artery stiffness reduces the arterial elasticity through the

recruitment of inelastic collagen fibers and causes hypertension [8]. Consequently, the heart

needs to work harder in order to pump blood to meet the demands of the body and thus,

increasing the chances of developing coronary events.

Pharmacological interventions are available to manage MetS risk factors such as

modulating blood lipids and lowering blood glucose. Pharmacological agents like atorvastatin

(e.g. Lipitor) and fenofibrate (e.g. Lipofen) are common medications for lowering blood lipids.

Atorvastatin reduces blood cholesterol by combinations of two major mechanisms 1) inhibiting 3-

hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity by up regulating the

apoB/E receptor which reduces synthesis of low density lipoprotein (LDL) and increases

removal of LDL from the plasma [9, 10]; and 2) reducing production of very low density

lipoprotein (VLDL) particles with less cholesterol ester but more triacylglycerol [11, 12].

Fenofibrate, decreases triglyceride levels while increasing HDL-cholesterol in plasma [13].

Thiazolidinediones (TZDs) (e.g. rosiglitazone), conversely, are used to restore insulin sensitivity

in type 2 diabetes patients by promoting free fatty acid ( FFA) uptake and triglyceride synthesis in

3

the subcutaneous tissue. TZDs also increases glucose uptake in the liver and skeletal muscle by

elevating the gene expression and translocation of glucose transporters GLUT1 and GLUT4 to

the cell surface [14]. Unfortunately, the use of these pharmacological agents, even for short term,

is associated with side effects [15]. Study shows that short-term use of rosiglitazone is associated

with an increased risk of myocardial infarction and death rate from cardiovascular disease [15],

increased stress on left ventricular wall and hence greater oxygen demand for myocardial cells

[16] , edema [16], and bone loss [17].

Because of the undesirable side effects associated with the use of pharmacological agents

to manage blood lipids and glucose, alternative treatments are being explored. Eating a well-

balanced diet is strongly encouraged as means of improving clinical parameters or maintaining

health. Fruits and vegetables are great sources of fiber, antioxidants and other bioactive

compounds that can lower glucose levels, modulate blood lipids, lower blood pressure, and

positively influence other MetS risk factors [18, 19]. The viscosity of the fibers found in fruits

and vegetables contributes to the cholesterol-lowering property. Fruits and vegetables have high

insoluble fiber content and are low in energy, fat and simple sugar, which contribute to reducing

the risk of obesity and hypertriglyceridemia [20]. Antioxidant, that are abundant in fruits and

vegetables have been shown to prevent the initiation or propagation of the oxidation process,

which protects against coronary heart disease and cancer [21]. Furthermore, bioactive

compounds such as vitamins and flavonoids in fruits and vegetables possess antioxidant and anti-

inflammatory properties, which helps in alleviating MetS risk factors such as obesity and insulin

resistance as well as prevent or delay other development of many chronic diseases [22].

One fruit that is rich in anti-oxidants, vitamins, and other bioactive compounds such as

flavonoids (e.g. quercetin and glucosylated xanthone) is mango. There has been increasing

interests in mango for many years in terms of health benefits [23]. Antioxidant vitamins

including vitamins A, C, E, are critical for prevention of oxidative damage to cell membranes and

4

thus degenerative diseases. Mango peel and pulp also contain carotenoids (provitamin A and

beta-carotene) and polyphenols such as quercetin, gallic acid, catechins which counteract free

radicals that are associated with various diseases. A bioactive compound mangiferin found in

mango bark, fruits, roots and leaves, has been used in India for the treatment of arteriosclerosis,

coronary heart disease and diabetes [24]. Studies have shown that mangiferin exhibits anti-

diabetic [25-27] , hypolipidemic and anti-atherogenic properties [26, 28, 29] by reducing plasma

total cholesterol, triglycerides, low density lipoprotein-cholesterol (LDL-C) and increasing levels

of high density lipoprotein (HDL-C) [26]. In addition, mangiferin has been shown to reduce the

production of reactive oxygen species and hence the risk of insulin resistance by restoring

mitochondrial redox homeostasis [30, 31] and also exhibit anti-inflammatory properties [32, 33].

However, to our knowledge, limited studies have been done comparing the effect of mango pulp

to mangiferin and rosiglitazone on clinical parameters associated with MS. How much mangiferin

contributes to the positive effect of mango pulp is not known. The specific aim of the study is to

compare the effects of freeze-dried mango pulp to that of mangiferin and rosiglitazone, a known

peroxisome proliferator activated receptor-ϒ (PPAR-ϒ) agonists, on reducing factors associated

with MetS.

Our null hypothesis is that there will be no difference between mangiferin, freeze-dried

mango, and rosiglitazone in

a) modulating body composition,

b) decreasing blood glucose and lipid concentration, and

c) modulating other clinical parameters in mice fed with high fat diet.

5

CHAPTER II

REVIEW OF LITERATURE

Metabolic syndrome prevalence

Metabolic syndrome (MetS) is a constellation of health issues including abdominal

obesity, hypertension, insulin insensitivity, dyslipidemia, resulting to an elevated risk for diabetes

and coronary heart disease. The presence of three of these five risk factors is considered

diagnosis of MS: 1) waist circumference >102 cm and 88 cm, for men and women, respectively

2) triglycerides >150 mg/dL (1.7mmol/L); 3) high density lipoprotein-cholesterol (HDL-C) <

40mg/dL (1.0mmol/L) and 50mg/dL (1.3mmol/L) for men and women, respectively; 4) systolic

or diastolic blood pressure >130 mmHg or 85 mmHg; respectively; and 5) glucose >100mg/dL.

Individuals with metabolic syndrome is strongly associated with prothrombotic and

proinflammatory states [1]. Obesity is the starting point for the development of metabolic

syndrome and obesity induced insulin resistance is a critical contributor to other chronic diseases

associated with MetS [34]. Therefore, the increasing prevalence and associated medical expenses

for both obesity and diabetes are of crucial concerns in the US and worldwide.

According to the National Health and Nutrition Examination Survey (NHANES) in 2007-

2008 [35], the percentage of obesity were 32.2 and 35.5 for adult men and women, respectively.

An estimated $147 billion were spent for medical bills associated with obesity in 2008 [36]. The

National Diabetes Fact Sheet 2011 released by Centers for Disease Control and Prevention

6

(CDC) [37] showed approximately 10.9 million or 26.9% of US citizens aged 65 years or older

were diagnosed with diabetes. Furthermore, diabetes affects approximately 215,000 children

younger than 20 years old. The total expenditure for diabetes was approximately $174 billion, in

which $116 billion was attributed to the direct medical costs of diabetes in the year 2010. On the

other hand, the indirect costs such as work loss, disability and premature mortality were

approximately $58 billion. Consequently, all the costs associated with obesity-related chronic

conditions imposed enormous negative impact on the nation’s productivity.

Pathophysiology of obesity

Obesity is a condition of excessive adipose tissues. Adipose tissue is a specialized type of

connective tissue composed of fat cells embedded in a collagenous structure in order to provide

support to the fat cells, capillaries and nerve fibers that penetrate the adipose tissues. The fat-

storing adipocytes are influenced by dietary intake and hormonal responses [38].

The amount of fat in adipose tissue normally stays constant even though continuous turn

over occurs. A healthy individual exhibits homeostatic control in terms of fat synthesis and

storage by regulating the balance between lipid breakdown and mobilization [39] . Obesity, on

the other hand, demonstrates abnormality in terms of lipid storage regulation in fat tissues. The

accumulation of fat in adipose tissues arises due to the transfer of plasma lipid to fat cells [39].

The lipoprotein bound in triglyceride like chylomicrons or VLDL are acted upon by the enzyme,

lipoprotein lipase, to release glycerol and fatty acids where the fatty acids will then enter into the

fat cells and are converted to triglyceride for storage [39]. Triglyceride (TG) storage is formed

from both fatty acids and α-glycerophosphate, in which glucose uptake and metabolism are

necessary in order to provide sources of α-glycerophosphate from glucose. [39].

When a person is energy deprived, the free fatty acids (FFA) are released from the TG

stores in the adipose tissues and bind to the albumin in circulation [39]. The release of the fatty

7

acids is activated by lipolytic hormones such as catecholamines (epinephrine and

norepinephrine), glucagon, and secretin [39]. When the hormones bind to the receptors on the

adipocytes surface, the enzyme adenyl cyclase is activated. The stimulation of the enzyme adenyl

cyclase increases the levels of cyclic-3’,5’-AMP (cAMP), which activates lipolytic lipase [39].

Furthermore, the presence of insulin exhibits anti-lipolytic properties through general

mechanisms including the inhibition of the enzyme adenyl cyclase, the activation of the cAMP

phosphodiesterase which leads to cAMP degradation, and the promotion of glucose uptake and

lipid storage [39]. When food intake exceeds energy expenditure, accumulation of fat happens by

expanding the existing fat cells (i.e. hypertrophy) or forming new fat cells (i.e. hyperplasia),

hence, obesity occurs if this situation persists [40].

Obesity is also strongly associated with inflammation, which was well demonstrated by

Hotamisligil and colleagues [41]. This group of researchers showed excessive expression of the

pro-inflammatory cytokine, tumor necrosis factor- alpha (TNF-α) in adipose tissue of obese

individuals. The connection between obesity and inflammation is further reinforced when

elevated plasma concentration of proinflammatory mediators such as TNF-α, interleukin-6 (IL-6),

C - reactive protein (CRP), and migration inhibitory factor (MIF), were found in obese people

[42-46]. Ghanim and colleagues [47] demonstrated that increased levels of proinflammatory

cytokines were found in adipose tissues of the obese individuals. Moreover, a significant increase

of the binding of nuclear factor-κB (NF- κB) and declined levels of the inhibitor of NF-- κB-

kappaB kinase beta (IĸBβ) were observed. Therefore, inflammatory processes take place in

obesity.

8

Insulin resistance

Since obese individuals have excess TG storage in adipose tissues, the risk of developing

insulin resistance increases. It has been demonstrated that obese individuals exhibit high rates of

breakdown and uptake of fatty acids [48]. FFAs taken up by the skeletal muscle and liver undergo

β-oxidation in the mitochondria or are stored as triglycerides [49]. When the skeletal muscle or

liver cells are saturated with products of β-oxidation, increased triglyceride synthesis occurs.

Metabolites of FA synthesis, diacylglycerol (DAG) and ceramide start to accumulate [50] . DAG

and ceramide up- regulate protein kinases like JUN N-terminal kinase 1 (JNK1) and the inhibitor

of nuclear factor-κB (NF- κB) kinase-β (IKKβ), which inhibit insulin action by increasing the

inhibitory serine phosphorylation of insulin receptor substrates (IRS) [51-53]. Phosphorylation of

IRS inhibits insulin signaling resulting to insulin resistance [54].

The expansion of adipose tissue in obese individuals leads to inadequate supply of

oxygen to the adipose tissues [6, 7, 55] , which activate JNK1, IKKβ and other genes involved in

endoplasmic reticulum (ER) stress and inflammation. Up-regulation of JNK and IKKβ activates

expressions of inflammatory pathway and genes, which is crucial for the activation, adhesion and

migration of macrophages to the adipose tissues [56]. The release of chemokine from adipose

tissue recruits macrophages in the adipose tissue [55, 57, 58]. Majority of the content in obese

adipose tissue macrophages (ATM) are pro-inflammatory macrophages which secrete TNF- α and

IL-6. Increased production of TNF- α and IL-6 stimulate serine phosphorylation of IRS-1and

prohibits the binding of IRS-1 with the insulin receptor, thus, inhibiting insulin signaling [59, 60].

Obesity is also associated with an accumulation of unfolded proteins in the endothelium

reticulum (ER). A protective response known as unfolded-protein response (UPR) functions as a

homeostatic restoration of ER [61, 62]. Inositol-requiring kinase 1(IRE1) is one of the stress

sensing mediator transmembrane proteins located in ER [61, 63, 64]. An elevated level of plasma

9

glucose often associated with obesity, induces the autophosphorylation of IRE1 and leads to

degradation of IĸB. Hence, NFĸB is secreted and an activation of pro-inflammatory response

occurs due to ER stress [65, 66]. Chronic elevation of circulating glucose activates both the JNK

and IKK-NFĸB signaling pathways, which up-regulates the pro-inflammatory genes and causes

impairment of insulin [62] .

Another consequence of obesity is ectopic accumulation of fat in muscle, which

contributes to mitochondrial dysfunction [67] . Mitochondrial dysfunction causes a decline in the

expressions of nuclear encoded genes which modulate the mitochondrial homeostasis like PPAR-

ϒ [68]. The elevated levels of intracellular FA metabolites like DAG and ceramide, increase

oxidative stress due to increased incidence of mitochondrial uncoupling, which leads to the

production of reactive oxygen species (ROS) [69]. ROS and oxidative stress, in return, activate

the protein kinases signaling cascades, serine phosphorylation of IRS-1 and thus, impairs insulin

sensitivity [30, 31].

Hyperinsulinemia, a condition in which excessive circulating insulin in plasma relative to

the plasma glucose contributes to insulin resistance. It has been shown that the presence of

insulin triggers the expressions of the main hepatic lipogenic transcription factor, SREBP-1c and

increased expressions of liver SREBP-1c was observed in obese rodents [70]. Consequently, the

rodents exhibited reduced insulin sensitivity due to the down regulation of mRNA for IRS-2,

which is a critical component insulin signaling [71]. Therefore, hyperinsulinemia causes hepatic

lipogenesis-steatosis as well as debilitating the ability of insulin to suppress hepatic glucose

output [71].

10

Atherosclerosis

A chronic high fat dietary intake increases the permeability of the endothelium. The

increased permeability is due to the accumulation of lipids and lipoproteins beneath the

endothelium. Peripheral monocytes and T-lymphocytes attach and adhere to the endothelial cells

through adhesive cell-surface glycoproteins while leukocytes attach to endothelium through

ligand-receptor mechanism, leading to inflammatory response [72]. Chemokine produced during

the inflammatory response bind to endothelial surface which attracts the leukocytes into the

subendothelial space. In addition to leukocytes, monocytes are converted to activated

macrophages and take up the oxidized low-density lipoprotein through the scavenger receptors

which can develop into foam cells. The accumulation of foam cells in the intima lead to

formation of fatty streak and atherosclerosis lesions. The persistence of inflammatory actions

adversely affects the artery wall [73]. Remodeling of the atherosclerostic lesions to a formation of

fibrous plaque may occur if inflammatory response continues. Consequently, the integrity of the

artery wall is disrupted, and atherosclerosis arises [73].

An increased level of circulating FFAs due to high fat diets is also a predominant

contributor the production of highly atherogenic, easily oxidized and less readily cleared, small

and dense low-density lipoproteins (LDL) [74]. In the liver, high fat diet leads to increase

synthesis of very-low-density lipoproteins (VLDL) and decrease triglyceride clearance [75]. The

declined triglyceride clearance is due to decreased activity of lipoprotein lipase and cholesterol

ester transfer protein (CETP), which mediates the transfer of triglyceride from VLDL to HDL in

exchange of cholesteryl esters. Reduced activity of hepatic lipoprotein lipase and CETP results to

an increase in small and dense LDL, hence increasing the likelihood of atherosclerosis [75].

Other factors such as elevated glucose can also contribute to an increase in

atherosclerotic lesion formation. A high concentration of circulating blood glucose which

determines the amount of the formation of advanced glycosylation end products (AGEs) among

11

long-lived proteins such as collagen in insulin-independent tissues [76] also contributes to

atherogenesis. As a result, the irreversible AGE-linked collagen leads to alteration of the

mechanical properties of the interstitial tissues of the arterial wall and thus arterial stiffness [77].

The endothelial cell function deteriorates because AGEs diminishes the generation of nitric oxide

but increases the production of oxidant species like peroxynitrate [78] . In addition, AGEs trigger

inflammatory responses and stress signaling, which increase the production of NF-κB, free

radicals, proinflammatory cytokines and vascular adhesion molecules [79, 80] . The elevated

concentration of proinflammatory and stress mediators weaken the integrity and the elasticity of

the endothelial cells through matrix metalloproteases (MMPs) [81], leading to arterial stiffness.

Moreover, MMPs also causes smooth muscle tone elevation, suppress endothelial flow-mediated

dilation, deteriorate vascular injury responses and functions of angiogenesis and enhance the

formation of atherosclerotic plaque, which are all contributors to coronary disease [82-84].

Hypertension

Blood pressure elevation is common among obese individuals. The increased production

of pro-inflammatory molecules like TNF-α and IL-6 in obese individuals are associated with

increased blood pressure [54]. As TNF α and IL-6 trigger serine phosphorylation of IRS-1, the

binding of IRS-1 with the insulin receptor is prevented, leading to inhibition of insulin signaling

[61, 62]. Consequently, the plasma concentration of insulin remains elevated which can promote

renal sodium reabsorption as insulin possess anti-natriuretic effects [85-87]. The renal sodium

retention increases salt sensitivity and intravascular fluid, leading to increased cardiac output and

peripheral resistance. Blood pressure increases accordingly and therefore, hypertension develops

[88] .

Insulin resistant individuals also possess high levels of plasma endothelin-1 [89], which

functions as a vasoconstrictor. Endothelin-1 activation results in the formation of inositol 1,4,5-

12

trisphosphate (IP3) which mobilizes calcium ions from sarcoplasmic reticulum (SR) , leading to

skeletal muscle vasoconstriction, increased blood pressure and thus hypertension [89].

The secretion of IL-6 by adipose tissues also triggers the activation of sympathetic

nervous system, followed by a rise in plasma angiotensionogen and angiotensin II, resulting to

hypertension development [90]. It has been shown that muscle sympathetic nervous activities

(MSNA) in obese patients are significantly higher than normal individuals [91]. As a result, the

elevation of resting heart rates in conjunction with baroreflex dysfunction contributes to

hypertension [92, 93] . Renin-angiotensin system (RAS) regulates blood pressure through

modulation of vascular tone and renal sodium reabsorption [94] and is influenced by food intake.

As a result, calorie overload causes increased expressions of gene for angiotensinogen

(angiotensin converting enzyme) and type I angiotensin in adipose tissue leading to increased

angiotensin II formation in adipocytes [94]. Subsequently, angiotensin II imposes inhibitory

effects to insulin action via increased concentration and activity of RhoA and reactive oxygen

species generation, leading to a decline in production of NO in endothelial cells and elevated

vasoconstriction [95] and therefore, resulting to hypertension.

13

Peroxisome proliferator activated receptor agonists

Thiazolidinedione (TZD) drugs are generally used to modulate plasma glucose levels

among type 2 diabetics. The common TZDs used in the United States are rosiglitazone (Avandia,

GlaxoSmithKline) and pioglitazone (Actos, Takeda) and the anti-diabetic actions are mediated

through the activation of peroxisome proliferator activated receptor-ϒ (PPAR-ϒ). There are three

isoforms for peroxisome proliferator activated receptor (PPAR)—PPAR-α, - γ and –δ. All the

PPAR isoforms are nuclear receptors and are activated by fatty acids and fatty acid-derived

eicosanoids [96].

The expressions of PPAR-α is mostly in tissues which are involved in fatty acid

catabolism like liver, skeletal muscle, heart, and kidney [97]. PPAR- γ is expressed

predominantly in adipose tissue, it is also found in vascular endothelium, pancreatic beta cells and

macrophages [98, 99]. PPAR- δ, on the other hand, is highly expressed in the brain, adipose

tissue and skin [100].

When TZDs bind to PPAR-ϒ, activation of PPAR-ϒ occurs and heterodimers of PPAR-

RXR (retinoid-X-receptors) are formed. The binding of the heterodimers to DNA-specific

sequences (peroxisome proliferator-response elements) promotes or deactivates the transcription

of target genes regulating glucose and lipid metabolism. Since PPAR isoforms exhibit sensitivity

and selectivity to ligands and the coactivator proteins, diverse sets of genes [101] involved in

fatty acid uptake and oxidation, inflammation, and vascular functions are modulated. The

regulation of the process in the uptake and storage of the fatty acid, glucose homeostasis and

inflammation are controlled by the genes encoding PPAR- γ. The activation of PPAR-α induces

oxidation of free fatty acids, promotes anti-inflammatory responses, and regulates genes that

modulate lipoprotein concentrations [102-104]. Whereas gene encoding for PPAR- δ is

responsible for the metabolism of fatty acid, inflammation and macrophage lipid homeostasis

[101]. Since rosiglitazone is an agonist for PPAR- γ, the mechanisms of actions of PPAR- γ in

14

promoting glucose and lipid metabolism will be the focus of discussion.

PPAR- γ, insulin sensitivity and dyslipidemia

The synthetic ligands for PPAR- γ have been demonstrated to be effective for treating

diabetes mellitus due to its ability to restore insulin sensitivity. The cellular actions of PPAR- γ

agonists include stimulation of adipocyte differentiation from the uptake of the FFA and hence fat

storage in subcutaneous rather than visceral adipose tissue, leading to a reduction of FFA levels

and FFA- induced insulin resistance. Moreover, when the expression of PPAR- γ increases,

translocation of the glucose transporters GLUT1 and GLUT4 to the cell surface increases.

Therefore, the plasma glucose concentration is reduced as increased rate of glucose uptake to the

liver and skeletal muscle cells occur [14]. Another mechanism of action of PPAR- γ agonists to

improve insulin sensitivity is by increasing the secretion of adiponectin [105] while reducing the

secretion of TNF- α [106]. The presence of adiponectin increases FA oxidation in the liver [107]

and muscle cells [108-110]. When circulating FFA declines, the number of macrophages

surrounding the adipose tissue decreases. Therefore, the inflammatory response triggered by

TNF- α from the macrophages is attenuated, which improves insulin resistance [59, 60].

In addition to increasing insulin sensitivity, the activation of PPAR- γ regulates lipid

homeostasis by promoting FA uptake and storage in adipose tissue, leading to adipose tissue mass

expansion. Gene expression of several receptors and proteins are also up-regulated by activation

of PPAR- γ. These proteins include 1) CD36, receptor important for increased uptake of oxidized

LDL [111]; 2) acyl-CoA synthetase, for triglyceride synthesis; 3) lipoprotein lipase, enzyme for

hydrolyzing triglyceride in chylomicron and VLDL and providing NEFA and monoacylglycerols

for utilization of tissues [112, 113]; 4) glycerol kinase (GyK), enzyme important for the

conversion of glycerol to glycerol- phosphate, compound involved in TG synthesis [114]; 5)

ABCA1, which is a membrane transporter responsible for excess cholesterol from extrahepatic

cells to be transported to the liver for bile acid synthesis or cholesterol recycling [115-118].

Additionally, activation of PPAR-ϒ reduces secretion of leptin [119, 120] leading to decreased

15

caloric intake [121-123] and TNF-α, which can improve insulin signaling pathway by increasing

the binding of IRS-1 to the insulin receptor [59, 60].

Adverse effects of Thiazolidinediones (TZDs)

The most common side effects of the use of TZDs include weight gain, edema, bone loss

and increased risks of death from cardiovascular disease. The next section will discuss studies

that demonstrate the side effects of TZDs particularly rosiglitazone.

Weight gain

Weight gain is the most common side effects of rosiglitazone use [124]. A mean

increased in weight of 1.9 kg and 2.9 kg was observed with the daily use of rosiglitazone (4 mg

and 8 mg, respectively) for 52 weeks [124] . In a 26-week study, the co-administration of

rosiglitazone (4-mg daily dose) with sulfonylurea or metformin both showed increased weight of

1.8 kg as compared to monotherapy of sulfonylurea or metformin [125]. The weight gain effect

was more prominent when rosiglitazone was co-administered with insulin. The increased weights

of 4.1kg and 5.4kg were observed when daily doses of rosiglitazone at 4- mg and 8- mg were

added to the insulin, respectively.

Administration of TZD alone or co-administration with metformin, sulfonylurea, or

insulin lead to weight gain as a result of fluid retention [124]. Plasma volume was shown to

significantly increase by 1.8 mL of water per kg body weight among the healthy individuals

receiving 8mg of rosiglitazone once daily when compared to placebo [124]. Consequently,

subjects receiving rosiglitazone had hemoglobin levels decreased by 0.8 -1.1 g/dL relative to

whether the drug was used as monotherapy or combination of both rosiglitazone with

sulfonylurea, metformin, or insulin [124].

16

Edema

With increased fluid retention, edema is also a common side effect of rosiglitazone.

Compared to placebo, edema occurred in 4.8 % vs. 1.3 % of the 9,900 type 2 diabetic patients

under Avandia medications [124]. Additionally, 3- 4 % of patients receiving the combination

therapy of rosiglitazone with metformin or sulfonylurea, also experienced edema compared to

only 1.1-2.2 % of the patients under metformin or sulfonylurea alone [124]. These findings

demonstrated that edema is an associated side effect with TZD drugs, irrespective of mono or

combined therapy with other anti-diabetic agents.

The pathogenesis of fluid retention leading to edema with the use of TZD is not fully

understood. Since the reciprocal relationship between plasma volume and renal sodium excretion

is well established, it is believed that edema is caused by the increased rates of sodium and water

retention [126]. The synergistic interaction between TZD and insulin induces arterial

vasodilatation, activates sodium reabsorption and increases extracellular volume, leading to pedal

edema [126]. Other possible contributors to edema include increased sympathetic nervous system

actions [127], alterations in transport of interstitial ion [128] and endothelial permeability [129],

as well PPAR-ϒ-induced regulation of vascular permeability growth factor [130].

Bone Loss

Animal and human studies have shown that rosiglitazone use can lead to bone loss

[17, 131, 132]. Rzonca and colleagues found that 6-month old, non-diabetic C57BL/6 mice

showed significant total bone mineral density reduction with the administration of rosiglitazone

of 20 µg/g body weight per day for 7 weeks. Bone volume, trabecular number and width were

reduced while trabecular spacing was increased. The researchers also found decreased gene

expressions of the osteoblasts transcription factor, runt-related transcription factor-2 (Runx2)

[132]. Similarly, in a study conducted by Afshan et.al.found that the administration of

17

rosiglitazone (25 µg/g body weight) daily for 28 days in 5-month old Swiss-Webster mice,

showed signs of bone loss. The signs of bone loss include increased marrow adipocytes, and

decreased in the ratio of osteoblasts to osteoclasts, rate of bone formation and the width of bone

wall. Furthermore, the gene expressions for osteoblasts transcription factors Runx2 and Osterix

also decreased in the cell cultures of marrow-derived mesenchymal progenitors [17].

One mechanism to explain bone loss associated with rosiglitazone is the modification

of common mesenchymal progenitors from osteoblast to adipocyte lineage, which leads to

increase formation of adipocytes, but a decline in osteoblasts [133, 134]. This reciprocal

relationship of marrow adipocytes and osteoblasts is due to their derivation from a common

mesenchymal progenitor [135-138]. The suppression of the osteoblast lineage causes reduced

expressions of Runx2 and Osterix genes, crucial transcription factors for osteoblastogenesis.

Consequently, the expressions for bone matrix proteins such as collagen, bone sialoprotein,

osteocalcin will be arrested, leading to bone loss [139, 140].

Increased risks and death from CVD

A meta-analysis of 42 clinical trials (24 to 52 weeks in duration) comparing the

effects of rosiglitazone with placebo or comparable oral anti-diabetic agents on cardiovascular

outcomes was conducted by Nissen and Wolski [15]. The odds ratio for risks of myocardial

infarction and death from cardiovascular causes in the rosiglitazone group, when compared to the

control group, were 1.43 (95% confidence interval [CI], 1.03 to 1.98; P=0.03) and 1.64 (95% CI,

0.98 to 2.74; P=0.06), respectively. This indicated that rosiglitazone was associated with

approximately 1.5 times risk of developing myocardial infarction and death from cardiovascular

diseases [15].

Rosiglitazone was also shown to affect the lipid profile. A mean increase of 18.6% in

LDL cholesterol among individuals on rosiglitazone (8-mg daily dosage for 26 weeks) as

18

compared to placebo was demonstrated by Khan et.al. [125]. This increase in LDL cholesterol

was a contributor to negative cardiovascular outcomes with rosiglitazone use [16]. Furthermore,

rosiglitazone and other TZD could cause congestive heart failure among vulnerable patients [16].

Congestive heart failure is strongly associated with increase in intravascular volume. The stress

level on the left ventricular heart elevates when the intravascular volume increases, leading to

increased oxygen demand for myocardial cells. Therefore, the chances of triggering ischemic

events are generally higher among susceptible patients [16].

Considering the use of rosiglitazone is associated with negative side effects, the

incorporation of fruits and vegetables which are rich sources of nutrients and bioactive

compounds is one alternative in preventing chronic conditions. A fruit such as mango, a rich

source of vitamins and polyphenolic compounds which have antioxidant properties, may help

prevent MetS risk factors.

Composition of mango

Typical composition of mango pulp is shown in Table 1. Mango flesh is rich in

various nutrients particularly vitamin C and carotenoids. Vitamin C is the predominant vitamin in

mangos and the amount declines with maturity or ripening process [141] . The quantity of vitamin

C present in the mango peel is about 1.5 times higher than in the mango pulp [141] . Vitamin C

has been well-documented to have antioxidant characteristics which helps in ROS elimination,

the maintenance of α-tocopherol and cofactor in the reduced state and preserving the enzymatic

activities , act as a substrate for the biosynthesis of oxalate and tartrate in resisting stress [142,

143], and the production of hormones, neurotrasmitters as well as collagen. Therefore,

degenerative diseases like cardiovascular disease, inflammation, cancer, arthritis, and the

weakening of the immune systems can be minimized by sufficient intakes of antioxidants such as

vitamin C.

19

The carotenoids contribute to the yellow or orange color of the ripe mango flesh

whereas unripe mangos do not possess carotenoids. The presence of carotenoids in mango makes

the fruit tastes sweet and less acidic [144]. Trans-ß-carotene has been reported to be highest

amount among all the carotenoids in the mango pulp [145] and the levels ß- carotene increases

from mature green to ripe [146]. ß- carotene is known to exhibit antioxidant property [145] and

prevents or delays the development of cardiovascular disease, cataracts, inflammatory, immune

disorders and muscular disease [147]. The vitamin A content in mango pulp is 1.17 mg/100g

[141, 148] and the total carotenoids fall within the range of 0.9-0.92 mg/100g [145].

Table 1: Typical nutrient composition of mango pulp [141]

Constituent Units Value per 100 grams

Macronutrients

Protein g 0.51

Fats g 0.27

Carbohydrates g 17.00

Total dietary fiber g 0.50

Minerals

Calcium mg 10

Iron mg 0.13

Magnesium mg 9.0

Phosphorus mg 11

Potassium mg 156

Vitamins

Vitamin C

(Total ascorbic acid)

mg 27.2

Total folate mcg 14

Vitamin A IU 3894

Vitamin E Alpha tocopherol equivalent

(ATE)

1.120

20

Polyphenolic compounds

Mango is also a rich source of polyphenolic compounds. The amount and type of

phenolic compounds in mango differs depending on the mango parts. Typical composition and

content of polyphenolic compounds in different parts of mango plant are presented in Table 2.

Table 2: Total phenolic and individual phenolic compound in mango pulp, peel, kernel and stem

bark [149-152]

Compound Mango pulp Mango peel Mango

kernel/seed

Mango stem

bark

Total phenolics

[mg (GAE) / 100g ]

ND 4066 112 10610

Individual polyphenolic compound (mg/kg)

Gallic acid

6.9 ND 0.60 0.24

Mangiferin

4.4 1690.4 0.42 71.4

Quercetin-3-O-

galactoside

22.1 82.0 ND ND

Quercetin-3-O-glucoside

16.0 557.7 ND ND

Anthocyanins

ND 0.2-0.5 ND ND

Tannin

ND ND 2.07 ND

ND= No data.

The most abundant flavanol found in mango is quercetin and it is responsible for the

diverse colors of fruits, vegetables and flowers [23]. It has been reported that quercetin is present

in unripe as well as mature mango fruits [152]. The predominant quercetin constituent in mango

pulp is found attached to galactose and glucose (Table 2). Wilkinson et.al [153] evaluated the

abilities of mango components such as quercetin, mangiferin, and norathyriol, which is an

aglycone derivative of mangiferin in modulating the transactivation of PPAR isoforms through

gene reporter assay. They observed that quercetin and norathyriol suppressed the transactivation

21

of all PPAR isoforms. PPAR nuclear receptors play crucial roles in diverse cellular and metabolic

processes. The modulation effects shown by quercetin and norathyriol suggested that these

compounds are able to modulate metabolic homeostasis and inflammatory processes, which

contribute greatly in improving metabolic syndrome risk factors.

Apart from flavonoids, phenolic acids such as gallic acid are also one of the major

constituents in mango pulp [154]. Gallic acid and ellagic acid, exist in free form or bound as

gallotannins or ellagitannins [23]. Gallic acid and ellagic acid contribute greatly to health as it has

been reported to exhibit antioxidant and anti-inflammatory [155, 156] It has been shown that the

content of gallic acid decreased significantly from mature green to advanced stage of fruit

maturity [157] . However, Kim and colleagues [157] observed the gallo-tannins increased in

Tommy Atkins mango cultivar during ripening, suggesting different cultivars, harvest locations

and climate might contribute to the different contents of polyphenolic compounds in mango.

Mangiferin

Figure 1: Mangiferin structure [23]

Mangiferin (Figure 1) is a C-glucosyl xanthone which is found in various parts of the

mango part. The content of mangiferin in mango stem bark is 71.4 g/kg, and it has become the

focus of researchers due to its abundance [158]. The standard aqueous extract of stem bark of

mango in pharmaceutical formulations in Cuba is under a brand named Vimang®. Vimang® has

a wide variety of polyphenolic compounds including the predominant xanthone constituent of

mangiferin, phenolic acids, phenolic esters and flavon-3-ols [158]. The amounts of mangiferin in

22

mango pulp, seed kernel and peel are reported to be 4.4 mg/kg [159], 42 mg/kg [160] , and 1690

mg/kg [149] , respectively. Studies on the health benefits of mangiferin as well as the stem bark

extract, Vimang® are discussed in the later section.

Factors affecting the contents of antioxidant compounds in mango

It has been demonstrated that different factors affect the antioxidant content of the

mango fruit [149, 157-160]. Cultivar as well as climate [161], ripening stage [162], and post-

harvest storage [163] affect the antioxidant content. Rocha Ribeiro et.al. [164] investigated the

amounts of total phenolics, total carotenoids, ß-carotene and total ascorbic of mango cultivars

grown in Brazil (Table 3).

Table 3: Total phenolics, total carotenoids, β-carotene, and total ascorbic acid in the pulp of four

mango cultivars [164-167].

Haden Uba Palmer Tommy Atkins

Total phenolics

(mg/100g) 48.4 208.7 62.1 128.2

Total carotenoids

(mg/100g) 1.91 ND 2.63 1.92

β-carotene

(µg/100g)

494.82 2220 661.27 1209-1405

Total ascorbic

acid

(mg/100g)

15.69 77.71 10.54 9.79

ND= No data

Similarly, Manthey and Perkins-Vaezie [168] also examined the amounts of

antioxidant compounds in mango pulp of five different mango cultivars relative to harvest dates

and locations.They observed that there were no consistent trends in ascorbic acid and β-carotene,

total phenolic contents relative to harvest locations. Interestingly, gallotannin contents in mango

pulp tend to have significantly higher in later harvest dates and this trend is consistent across

23

different harvest locations.

Mercadante and Rodriguez-Amaya [146] found that mango of the same cultivar have

different carotenoid compositions if the mango are grown in different climate regions. For

example, Tommy Atkins mango grown in hot area such as Mato Grosso, Brazil had higher

content of carotenoid compared to Tommy Atkins mango grown in moderate area like Sao Paulo.

Similarly, Keitt mango grown in hot area like Bahia, Brazil had twice as much as β-carotene

content compared to Keitt mango grown in moderate area like Sao Paulo, Brazil.

Ripening involves changes in cellular constituents and accelerates catabolic

processes, leading to changes in terms of color and firmness of the fruit [169]. Palafox-Carlos

et.al [162] examined the relationship between the total phenolic content and antioxidant

properties in ‘Ataulfo’ mango and the ripening stages. The ripening stage of mango is determined

by the yellow surface area in mango and it is categorized to 4 stages (RS1 to RS4) with the

percentage of yellow surface ranged from RS1, 0-10%; RS2, 11-40%; RS3, 41-70%; and RS4,

71-100%. They found that mangos from RS2 and RS3 possessed the highest phenolic

composition and antioxidant activities. Mercadante and Rodriguez-Amaya [146] assessed the

correlation between carotenoid contents and ripening stage in Keitt and Tommy Atkins cultivar.

Results showed total carotenoid increased from 12.3 to 38.0 µg/g and 17.0 to 51.2 µg/g in Keitt

and Tommy Atkins cultivar, respectively from mature-green to ripe stage.

Moreover, Gil et.al. [170] studied the changes in antioxidant compounds in mango

pulp during ripening by nuclear magnetic resonance spectroscopy (NMRS). They found that

peaks were observed for isoleucine, valine and leucine aliphatic in NMR spectra, suggesting a

common biosynthetic pathway for β-carotene. The content of β-carotene in mango pulp increases

with ripening (increased from 2.0 µg/g to 5.8 µg/g during ripening in Tommy Atkins mango

pulp). Conversely, in the aromatic region of the mango pulps’ spectra, it was found that shikimic

24

acid diminished gradually with fruit maturity with concomitant gradual increase of other

metabolites such as niacin and phenylalanine in the early ripening stage. The formation of

polyphenolic compounds was observed at the final stage of ripening.

One possible reason for explaining the changes of phenolic compounds and

associated antioxidant capacity during the ripening process is the increased cellular activity when

the fruit is maturing. The high cellular activity is accompanied with metabolism of biomolecules,

leading to increased demand for energy in fruits to support physiological cellular activities.

Hence, free radicals and ROS might be generated from the respiratory system, and activation of

the antioxidant mechanisms occurs to counteract the oxidative stress [162].

Hymavathi and Khader [163] studied the effects of storage duration and packaging of

the mango powders on the total carotene, β-carotene, ascorbic acid, and sugar content. They

found that the loss of total carotene, β-carotene and ascorbic acid is strongly associated with the

increased storage duration. In addition, packaging if permeable to moisture and air could

contribute to loss of ascorbic acid content in the mango powder. The total sugar content in the

mango powder increased with storage duration due to the conversion of non-reducing sugars to

reducing sugars.

Anti-diabetic and hypolipidemic effect of mangiferin

A few studies have demonstrated hypoglycemic and hypolipidemic effects of

mangiferin. Ichiki, et.al. [27] investigated the effects of oral administration of mangiferin (10

mg/kg, 30 mg/kg and 90 mg/kg) in animal model of non-insulin dependent diabetes mellitus

(NIDDM), KK-Ay mice. The treatment groups in this study were: KK-Ay mice as control group;

mice received 3 doses of mangiferin (10 mg/kg, 30 mg/kg and 90 mg/kg) respectively; mice

received mangiferin glucoside--mangiferin-7-O-β-glucoside (10 mg/kg, 30 mg/kg and 90 mg/kg),

respectively; and mice received tolbutamine (50 mg/kg), an anti-diabetic drug. They found that

25

mangiferin (30 mg/kg and 90 mg/kg) reduced blood glucose compared to mice with tolbutamide

treatment. Mangiferin at 90mg/kg showed more pronounced blood lowering property 7 hours

after the administration compared to tolbutamide.

Muruganandan and colleagues [26] examined the effects of intraperitoneal

administration of mangiferin (10 and 20 mg/kg) once daily for 28 days on diabetics and

atherogenesis in streptozotocin (STZ) - induced diabetic Wistar male rats. The five treatment

groups in this study were: normal rats received neither STZ nor drug; negative control was STZ-

induced diabetic rats; positive control rats with daily intraperitoneal administration of insulin

(6U/kg, i.p.) and rats given mangiferin 10 and 20 mg/kg, respectively. Blood was collected from

the fasted rats an hour later after the last dose of mangiferin and insulin administration to examine

blood glucose, plasma total cholesterol, HDL-cholesterol and triglycerides. Both doses of

mangiferin significantly reduced plasma glucose concentrations compared to the negative control.

Mangiferin also improved lipid profile by reducing plasma total cholesterol, triglycerides and

LDL-C with a concomitant increase in HDL-C in diabetic rats [26]. They suggested that the

positive effect of mangiferin on glucose can be attributed to the decrease in triglyceride, which

can be explained by Randle’s glucose fatty acid cycle. Randle’s glucose fatty acid cycle [171]

states that the elevation of plasma triglycerides increases availability of FFA for oxidation, which

then causes an impairment in insulin secretion that affect glucose metabolism and utilization,

resulting in hyperglycemia. Therefore, mangiferin is believed to ameliorate hyperglycemia by

promoting oxidation and glucose utilization [171]. The second objective of this study was to

assess the effects of mangiferin (10 and 20 mg/kg, i.p.) on oral glucose tolerance tests (OGTT) in

glucose-loaded normal mice compared to control normal mice with saline (1ml/kg i.p.) for 14

days. The last dose of mangiferin and saline were given 30 minutes prior the glucose injection to

the fasted mice. They found that the mice treated with mangiferin showed improved glucose

tolerance compared to the control group, hence mangiferin exhibited antihyperglycemic property

26

[26].

Miura et.al. [172] studied the effects of 2 weeks oral administration of mangiferin (30

mg/kg) combined with exercise on lipid parameters in KK-Ay mice, an animal model of type 2

diabetes. Mice were given food and deionized water ad libitum and the four treatment groups in

this study were: control, mangiferin (30 mg/kg), daily exercise on motorized treadmill for 120

minutes, and mangiferin combined with exercise. Mice given mangiferin 30 minutes before

subjected to exercise showed significant decrease in blood cholesterol and triglyceride levels

compared to the control diabetic mice. In addition, Guo and colleagues [29] also examined the

effects of mangiferin ( 50 mg/kg and 150 mg/kg ) in hamsters fed high fat diet for 8 weeks. The

four treatment groups in this study were: control diet (13.9% fat), high fat (HF) diet, and HF + 50

mg/kg mangiferin and HF + 150 mg/kg of mangiferin respectively. Hamsters that received

mangiferin showed significantly lower plasma triglyceride and FFA compared to the HF group.

Furthermore, mangiferin treatment up-regulated liver mRNA expression of proteins involved in

FA oxidation such as peroxisome PPAR-α, fatty acid translocase (CD36) and carnitine

palmitoyltransferase 1 (CPT-1). Down-regulation of hepatic mRNA expression of proteins

involved in FA synthesis such as acetyl coA carboxylase (ACC) and acyl-coA:diacylglycerol

acyltransferase 2 (DGAT-2) were also observed in mangiferin groups but not the HF group.

Sellamuthu and colleagues [173] examined the effects of daily oral administration of

mangiferin (40 mg/kg) for 30 days on STZ-induced diabetic rats. The four treatment groups in

this study were normal rats (control); diabetic rats (negative control); diabetic rats that received

daily oral administration of mangiferin (40 mg/kg) and diabetic rats given oral administration of

gilbenclamide (600 µg/kg), an anti-diabetic drug. The oral administration of glucose solution

during glucose tolerance test showed diabetic rats that received mangiferin and gilbenclamide

exhibited significantly lower plasma glucose, glycosylated hemoglobin and demonstrated

improved glucose tolerance test similar to normal glucose-loaded rats. Additionally, mangiferin

27

stimulated glucose utilization by increasing the enzymatic activities of hexokinase, an enzyme

involved in glycogen synthesis. The gluconeogenic pathway was also inhibited by mangiferin

through the suppression of enzymatic activities of glucose-6-phosphate (G6P) and fructose-1,6-

bisphosphate (F-1,6-BP). Mangiferin also restored glycogen levels similar to control normal rats

by activating glycogen synthase. Therefore, mangiferin showed anti-diabetic activity and restored

glucose homeostasis comparable to normal glucose loaded rats.

Antioxidant property of mangiferin

Mangiferin and its antioxidant property have been shown in scientific literature. It has

been well-documented that diabetes mellitus leads to ROS generation and subsequent oxidative

damage to the heart and kidney [174]. The increased plasma glucose produces free radicals which

induces cell death [175]. Muruganandan and colleagues [28] investigated the daily intraperitoneal

administration of mangiferin (10 mg/kg and 20 mg/kg) for 28 days on oxidative stress in STZ-

induced diabetic Wistar rats. The five treatment groups in this study were normal rats (control),

STZ-induced diabetic rats, STZ-induced diabetic rats given insulin (6 U/kg, i.p.), STZ-induced

diabetic rats given 10 mg/kg mangiferin and STZ-induced diabetic rats given 20 mg/kg

mangiferin . Blood samples were collected to determine glycosylated hemoglobin and serum

creatine phosphokinase (CPK) while the heart and kidney samples were used to examine the

antioxidant enzymes and histology. Compared to diabetic rats given insulin, mice that received

both doses of mangiferin had lower % glycosylated hemoglobin and serum CPK, a biomarker for

myocardial infarction. Moreover, mangiferin treatment also showed improvement in cardiac and

renal antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), reduced

cardiac and tubular degenerative signs in histological evidence when compared to diabetic rats

given the insulin treatment. Hence, mangiferin demonstrated anti-diabetic and attenuation of

oxidative stress in cardiac and kidney cells.

28

Prabu et.al. [176] studied the antioxidant potential of intraperitoneal administration

of mangiferin (100mg/kg b.w. suspended in 2ml of dimethyl sulphoxide (DMSO) for 28 days in

isoproterenol (ISPH)-induced myocardial infarctions (MI) in Wistar rats. The four experimental

groups in this study were; control rats given DMSO, ISPH-induced myocardial infarction rats,

normal rats given mangiferin, and normal rats pretreated with mangiferin for 28 days followed by

ISPH-induced MI. Serum was collected to examine the levels of enzyme associated with

myocardial infarction, antioxidant enzymes and non-enzyme antioxidants. Myocardial infarction

causes increased concentrations of lactate dehydrogenase (LDH), creatine phosphokinase

isoenzymes (CK-MB), uric acid and decreased plasma iron binding capacity. Rats pre-treated

with mangiferin (100 mg/kg) showed significant decrease in LDH, CK-MB and uric acid.

Significant increases in plasma iron binding activity, cardiac antioxidant enzymes such as

glutathione peroxidase (GPX) and glutathione transferase (GST), extracellular antioxidant such as

ceruloplasmin, vitamin C and E in rats pre-treated with mangiferin compared to ISPH-induced MI

rats were observed. On the other hand, rats treated with mangiferin alone showed comparable

results to control rats in LDH, CK-MB, uric acid, iron binding capacity, GPX, GST,

ceruloplasmin, vitamin C and E. Therefore, mangiferin exhibited cardiac protection by reducing

biomarkers of myocardial infarction while scavenging free radicals by increasing the antioxidant

enzymes.

Moreover, antioxidants are also important to prevent the development of

atherosclerosis as ROS has been shown to oxidize LDL, which can lead to atherosclerosis [177,

178]. Gilberto et.al [179] examined the effects of oral administration of Vimang® (250mg/kg)

and mangiferin (40mg/kg) for 7 days in LDL receptor knockout (LDLr-/-

) mice. The four

experimental groups in this study were; LDLr-/-

mice given distilled water as negative control,

C57BL/6 mice given distilled water as positive control, LDLr-/-

mice given Vimang®, and LDLr-/-

mice given mangiferin respectively. LDLr-/-

mice are susceptible to oxidative stress associated

29

mitochondrial dysfunction. Hence, substantial amount of ROS accumulate in the mitochondria of

LDLr-/-

mice, causing calcium-induced membrane permeability transition (MPT), a condition that

can lead to cell death. The mitochondria of mice that received Vimang® and mangiferin

treatment showed decreased amount of ROS production, efflux of calcium ions and MPT

incidences when compared to negative control. Additionally, LDLr-/-

mice also possessed high

cholesterol levels, leading to high consumption of NADPH reducing equivalents, which are

mitochondrial antioxidant defense compounds. Mice receiving Vimang® and mangiferin

treatment reduced the dissipation of NADPH, leading to increased antioxidant defense ability

when compared to negative control. Thus, Vimang® and mangiferin protected mitonchondria

from oxidative stress.

In addition to the mitochondria, ROS also affect macrophages [180]. Sanchez et.al.

[151] compared the antioxidant properties of oral administration of Vimang® (50 mg/kg, 110

mg/kg, 250 mg/kg) , mangiferin (50 mg/kg), vitamin C (100mg/kg), vitamin E (100mg/kg) and β-

carotene (50 mg/kg) against 12-O-tetradecanoylphorbol-13-accetate (TPA)-induced oxidative

deterioration in serum and over production of ROS in peritoneal macrophages in mice.

Vimang® at 250 mg/kg protected plasma antioxidant enzymes such as SOD and GPX from

depletion while mangiferin did not show effects in preserving serum antioxidant enzymes. TPA-

induced oxidative damaged mice that received Vimang® (any of the doses used) or mangiferin

had approximately 70% or 44%, respectively, lower ROS in peritoneal macrophages compared to

negative control mice. The concentration of H2O2 is a measurement of production of ROS by

peritoneal macrophages. Vimang® showed dose-dependent effect on decreasing the production

of H2O2 while mangiferin also reduced the production of H2O2 by 40% compared to the negative

control. Hence, both Vimang® and mangiferin showed potent antioxidant properties. However,

Vimang® has more pronounced effect compared to mangiferin, suggesting synergistic effects of

the polyphenolic compounds.

30

Anti-inflammatory effect of mangiferin

Nitric oxide (NO) and prostanoids are the main chemical mediators in inflammatory

processes. The synthesis of these molecules is catalyzed by inducible isoforms of NO and

cyclooxygenase (cycloocygenase-2). The development of hypertension has been well reported to

associate with the alterations of the expressions and functions of isoforms of NO and

cycloocygenase-2. Beltran et.al [33] investigated the effects of incubation of Vimang® (0.1

mg/ml, 0.2 mg/ml and 0.5 mg/ml) and mangiferin (0.025mg/ml) with interleukin-1β (IL-β) on the

expressions of pro-inflammatory enzymes such as cyclooxygenase and nitric oxide synthase

(iNOS) in rats’ vascular smooth muscle cell cultures. All doses of Vimang® and mangiferin

inhibited the expressions of iNOS; with Vimang® showing dose dependent decrease compared to

cell culture incubated with IL-β (negative control). Vimang® (0.2 mg/ml and 0.5 mg/ml) and

mangiferin suppressed the expressions of cycloocygenase-2 compared to negative control, which

was the cell culture of rats’ vascular smooth muscle incubated with IL-β. The inhibition of the

expressions of cyclooxygenase-2 and iNOS showed both Vimang® and mangiferin help in

preventing inflammatory conditions [181, 182].

Garrido and colleagues also examined [183] the effects of oral administration of

Vimang® (50 mg/kg, 100 mg/kg and 200 mg/kg) before topical application of arachdonic acid

(AA)-induced inflammation on the ears of mice. All doses of Vimang® ameliorated the AA-

induced inflammation of ear edema. Long lasting inflammatory response could be induced by

phorbol esters such as phorbol myristate acetate (PMA), leading to increased production of

prostanoid and influx of cellular cells like neutrophils [184] . The induction of neutrophil

biomarkers such as enzyme myeloperoxidase (MPO) was suppressed by all doses of Vimang®.

Additionally, Vimang® also showed dose-dependent inhibitory effects on production of serum

TNF-α. The addition of lipopolysaccharide-interferon-ϒ (LPS-IFN-ϒ) to macrophage cell lines

leads to activation of two diverge arachidonic acid metabolic pathways (i.e. cyclooxygenase and

31

lipooxygenase pathways). The effects on these pathways can be determined by measuring

prostaglandin (PGE2) and leukoterines (LTB4) respectively. Vimang® also showed inhibitory

effects on PGE2, LTB4 and human recombinant synovial PLA2, a proinflammatory protein [185].

Taken together, Vimang® exhibits anti-inflammatory effects and could be classifired as a dual-

inhibitor considering its suppression effects on two distinct arachidonic acid pathways.

Therefore mangiferin, being the predominant constituent in Vimang®, demonstrated anti-

inflammatory property by suppressing the inflammatory mediators, inflammatory cells as well as

inflammatory pathways.

32

CHAPTER III

METHODOLOGY

Animal care

Forty two-month old C57BL/6 male mice were purchased from Charles River

Laboratories (Wilmington, MA). Animal handling and procedures were approved by the

Institutional Animal Care and Use Committee at Oklahoma State University. Mice were

acclimated for three days and were fed with standardized powdered rodent diet (AIN93M) [186].

After acclimation, mice were weighed and randomly divided into five dietary treatment groups

(n=8/group) balanced by initial body weight. Mice were maintained in a 12:12 hour light dark

cycle in a temperature controlled room.

Dietary treatments

Mice were randomly divided into five dietary treatment groups (n=8 mice/ group): (1)

control (AIN-93M), 10 % calories from fat ; (2) high fat ( HF) diet, 60 % calories from fat; (3)

HF with rosiglitazone (50 mg/kg diet) ; (4) HF diet with 1% (w/w) freeze dried mango pulp ; and

(5) HF diet with mangiferin (0.044 mg/kg diet) . The AIN-93M diet was purchased from Harlan

Teklad (Indianapolis, IN) and contained 75.8 % carbohydrate, 9.5 % fat, and 14.7 % protein by

calories [186]. The high fat diet was based on the formulation of Molnar et al. [187] and

contained 27.7 % carbohydrate, 58.9 % fat, and 13.4 % protein by calories. Ripe Tommy Atkins

33

variety mangos were purchased from a local grocery store, freeze dried, grounded and analyzed

for its nutrient composition (Table 4) and incorporated at 1% (w/w) dose to the HF diets. The

dose of mangiferin (42.4 mg/ 100 g) is equivalent to the approximate mangiferin content of the

1% freeze-dried mango [188]. All high fat diets were adjusted to have the same macronutrient

composition, as well as calcium and phosphorus. The composition of the experimental diets is

shown in Table 5.

Glucose tolerance test

Glucose tolerance tests were performed after 30 and 60 days of dietary treatment (n=8 mice/

group). Mice were fasted overnight but had access to deionized water. A drop of blood from the

tail was used to determine baseline blood glucose concentrations using AlphaTrak™ glucometer

(Abbott Laboratories, North Chicago, IL). Blood glucose concentrations were measured at 0, 5,

15, 30, 60 and 120 minutes after intraperitoneal injection with 20% glucose solution (2 g/kg body

weight). The trapezoidal rule was used to calculate area under the curve [189].

Necropsy and tissue processing

After two months of dietary treatment, mice were weighed and fasted for 12 hours before

the necropsy. Mice were weighed and injected with ketamine/xylazine (10:1) cocktail (0.15 ml/10

g of body weight) at necropsy. Body composition was determined using a GE Lunar Piximus

whole body dual-energy X-ray absorptiometer (Fitchburg, WI). Blood was collected from the

carotid artery into ethylenediaminetetraacetic acid (EDTA) coated tubes. Plasma was obtained by

centrifugation of whole blood for 20 minutes at 1300 g and was stored at -80 °C until further

analyses. Liver, spleen and kidney were weighed, snap-frozen in liquid nitrogen and stored at -

80°C. Portion of liver and white adipose tissues were fixed in 10% neutral buffer formalin until

further analyses.

34

Clinical analyses

The BioLis24i chemical analyzer (Carolina Liquid Chemistries Corporation™, Brea,

CA) was used to determine plasma concentrations of glucose, frustosamine, whole blood

glycosylated hemoglobin (HbA1c), total cholesterol, HDL-C, triglycerides and non-esterified

fatty acids (NEFA). All reagent kits were purchased from Carolina Liquid Chemistries

Corporation™ (Brea, CA) except for NEFA (Wako Diagnostics, Richmond, VA).

The determination of plasma glucose concentrations is based on the reaction of glucose

with adenosine triphosphate (ATP) in the presence of hexokinase and magnesium where glucose-

6 phosphate and adenosine diphosphate (ADP) are produced. Glucose-6-phosphate

dehydrogenase oxidizes glucose-6-phosphate and NAD+ to form 6-phosphogluconate and NADH.

The amount of NADH produced absorbs efficiently at 340 nm, which is directly proportional to

the amount of glucose in the plasma (Carolina Liquid Chemistries Corporation™, Brea, CA).

The measurement of fructosamine utilizes a colorimetric method using nitroblue

tetrazolium (NBT). Fructosamine are reductants under alkaline conditions. This property forms

the basis for the NBT procedure in which the dye NBT is reduced to formazane which is then

measured spectrophotometrically at 505 nm wavelength. The rate of formation of formazane is

directly proportional to the fructosamine concentration (Carolina Liquid Chemistries

Corporation™, Brea, CA).

The measurement of whole blood glycosylated hemoglobin (HbA1c) utilizes the

interaction of antigen and antibody. Total hemoglobin and HbA1c have the same unspecific

absorption rate to latex particles, latex-HbA1c-mouse anti human HbA1c antibody complex is

formed after addition of mouse anti human HbA1c monoclonal antibody. Agglutination is formed

when monoclonal antibody interacts with goat anti-mouse IgG polyclonal antibody. The amount

of agglutination is proportional to the amount of HbA1c absorbed onto the surface of the latex

35

and the absorbance is measured at 660 nm. The HbA1c value is determined from a curve of

calibration (Carolina Liquid Chemistries Corporation™, Brea, CA).

In determining the total cholesterol, esterified cholesterol is hydrolyzed by cholesterol

esterase to free cholesterol and free fatty acids. The total cholesterol is oxidized by cholesterol

esterase producing hydrogen peroxide (H2O2). Peroxidase catalyzed the reaction among H2O2,p-

hydroxybenzoic acid and 4-aminoantipyrine to form the red colored complex —quinoneimine ,

which absorbs strongly at 500 nm wavelength. The amount of chromogen released is directly

proportional to the total cholesterol concentration (Carolina Liquid Chemistries Corporation™,

Brea, CA).

The measurement of HDL- cholesterol assay utilizes a unique reagent which solubilizes

HDL- lipoproteins. HDL cholesterol reacts with cholesterol esterase and oxidase to form colored

chromogen, which is directly proportional to the HDL cholesterol concentration, which absorbs

bichromatically at 600 nm (Carolina Liquid Chemistries Corporation™, Brea, CA).

Plasma triglyceride was hydrolyzed by lipase to release glycerol and free fatty acids.

Glycerol is phosphorylated to glycerol-1-phosphate while ATP is converted to ADP in the

presence of ATP and glycerol kinase. Glycerol-1-phosphate is oxidized by glycerol phosphate

oxidase and H2O2 produce. Red colored quinoneimine was formed in the reaction of H2O2, with

p-chlorophenol and 4-aminoantipyrine, catalyzed by peroxidase. The complex absorbed

bichromatically at 505/692 nm, representing the triglyceride concentration (Carolina Liquid

Chemistries Corporation™, Brea, CA).

The conversion of NEFA (non-esterified fatty acid) to thiol esters of CoA happens in the

presence of ATP and CoA and is catalyzed by acyl-CoA synthetase. Acyl-CoA is oxidized to

produce H2O2 in the presence of acyl-CoA oxidase and peroxidase, allowing the condensation of

3-methyl-N-ethyl-N-(β-hydroxyethyl)-aniline with 4-aminoantipyrine and the formation of purple

colored complex which absorbs strongly at 550 nm (Wako Diagnostics, Richmond, VA).

36

Plasma insulin

The determination of plasma concentration of insulin was carried out by the enzyme

linked immunosorbent assay (ELISA) kits from Millipore ™ Corporation (Billerica, MA) using

microtiter plates that were pre-coated with anti-mouse insulin monoclonal antibodies. A second

biotinylated anti-mouse polyclonal antibody was added to the wells to capture the analytes in the

samples. Unbound materials from samples were then washed off and horseradish peroxidase was

added on to the immobilized biotinylated antibodies. Enzymatic activity of horseradish

peroxidase was monitored using 3, 3’, 5, 5’-tetramethylbenzidine as a substrate and the

measurement is done using a microplate reader at 450nm (Biotek Synergy HT, Winooski, VT).

The captured mouse insulin in the plasma is directly proportional to the increase in absorbance at

450nm/590nm. The concentration of insulin was calculated from a standard curve (Millipore ™

Corporation, Billerica, MA).

Liver and fecal total lipids

Approximately 0.2 gram of the liver powder sample was weighed, transferred into a 50

mL centrifuge tube, and extracted overnight with 25 mL of methanol:chloroform (1:2) solution.

Fecal samples were dried, ground, and approximately 1 gram of fecal powder was weighed, and

extracted with 10 mL methanol:chloroform (1:2) solution. Liver and fecal total lipid was

determined based on procedure of Folch et al.[190]. Sulfuric acid (0.05%) was added to the

methanol:chloroform solution, vortexed, and allowed to be separated into two phases. The

aqueous layer was aspirated off after the two phases were separated. The organic solution was

poured into pre-weighed aluminum pan and evaporated to dryness under the fume hood. After all

the organic solvent has evaporated, pans were again dried in the oven at 100ºC, cooled, and

weighed. The amount of liver lipid was expressed as lipid weight (in milligram) per gram of liver

sample while fecal lipid is expressed as milligram lipid per gram of fecal samples.

37

Liver and adipose tissue histology

Liver and adipose tissues were fixed in 10% neutral buffered formalin. Tissues were

trimmed and processed in Thermo Scientific Shandon-Citadel 1000 tissue processor (Austin,

TX). Tissues were then embedded in Leica EG1160 paraffin embedding machine (Buffalo Grove,

IL). 7µm thicknesses of the paraffin-embedded tissue block were sectioned using the high

performance Leica RM 2165 microtomes (Buffalo Grove, IL) and mounted onto glass

microscopic slides. Slides were air-dried and stained with hematoxylin and eosin (H&E). The

slides were ready to view under Nikon Eclipse TE 2000-U Inverted Microscope (Melville, NY) at

10x magnification. The effects of different dietary treatments on white adipose cell sizes and the

degree of steatosis in liver tissues were evaluated.

Statistical Analyses

Statistical analyses involved the computation of least square means and standard

deviation (SD) for each of the treatment group using SAS software version 9.2 (SAS Institute,

Cary, NC). Analysis of variance and least square means were calculated using the general linear

model procedure and the means were compared using Fisher’s least significant difference for

groups’ comparisons. Significant differences were considered when P < 0.05.

38

Table 4: Nutrient composition of freeze-dried Tommy Atkins mango used in the study1

Nutrients Content/100g

Carbohydrates (g) 85.8

Dietary fiber (g) 12.9

Protein (g) 5.01

Moisture (g) 4.52

Calcium (mg) 79.2

Phosphorus (mg) 123

Fat (g) 1.75

Calories (kcal/100g) 379

Calories from Total Fat (kcal/100g) 15.8

1 Nutrient composition was analyzed by NP Analytical Laboratories (St. Louis, MO)

39

Table 5: Composition of the experimental diets

Ingredient Normal diet

(AIN-93M)2

High fat

diet3,4,5

High fat diet +

1% mango

amount (g/kg diet)

Mango pulp1

Carbohydrate

Total

Cornstarch

Sucrose

Dextrinized Cornstarch

Mango (85.8%)

Protein

Total

Casein

Mango (5.01%)

Fat

Total

Soybean Oil

Lard

Mango (1.75%)

Fiber

Total

Cellulose

Mango (12.9%)

Vitamin Mix (AIN-93VX)

Mineral Mix

Total

Mineral Mix (Ca-P-Def)

Calcium Carbonate

Calcium from mango (0.0792%)

Sodium phosphate, dibasic (NaHPO4)

Potassium phosphate, monobasic

Phosphorus from mango (0.123%)

Sucrose

Choline Bitartrate

L-cysteine

Tert-butylhydroquinone

Kcal/100g

0

721

465.69

100

155

140

140

0

40

40

0

50

50

0

10

35

13.4

12.5

0

5.6

2.4

1.1

2.5

1.8

0.008

367

0

370

0

270

100

180

180

0

350

40

310

50

50

0

10

35

13.4

12.5

0

4.8

2.0

2.3

2.5

1.8

0.008

559

10

370

0

270

91.42

8.58

180

179.5

0.5

350

39.82

310

0.18

50

48.71

1.29

10

35

13.4

12.48

0.0079

4.734

2.036

0.0123

2.35

2.5

1.8

0.008

547

40

1

Tommy Atkins variety; purchased from local grocery store, peeled, and pulp was freeze-dried. 2

AIN-93M contained 75.8 %carbohydrate, 9.5 %fat, and 14.7 %protein by calories [186]. 3

High fat diet formulation containing 27.7 %carbohydrate, 58.9 %fat, and 13.4 %protein by

calories [187]. 4

Rosiglitazone was from Cayman Chemical Company (Ann Arbor, MI) and added to the HF diets

at a dose of 50 mg/kg diet. 5 Mangiferin was from Sigma-Aldrich Co., LLC (St. Louis, MO) and added to the HF diets at a

dose of 0.044 mg/kg diet. 6

Analyzed by NP Analytical Laboratories (St. Louis, MO)

41

.CHAPTER IV

FINDINGS

Food intake, body and tissue weights

Body weights were similar at the initiation of treatment (Table 6 and Figure 2). However,

significant differences in body weights were observed starting at week one of dietary treatment

(Figure 2). All HF diet groups had similar body weight and were significantly higher than the

control group during the first two weeks of treatment. Starting at week 3, HF + rosiglitazone and

HF + 1% mango groups had slower weight gain than HF and HF + mangiferin which was

sustained until the end of the eight week treatment period.

HF-fed mice were fed ad libitum for the entire study duration. Mice receiving the control

diet were also fed ad libitum during the first four weeks of the dietary treatment but the amount of

food given to them for the last four weeks was controlled to be similar to the average intake of the

HF-fed groups. This allowed us to make sure the change in body weights in the control group was

not due to the amount of food eaten. The average food intake per day for the entire eight week

treatment period was highest in the control group followed by the HF + rosiglitazone group. HF,

HF + mangiferin, and HF + 1% mango groups had statistically similar food intake. Caloric intake

was highest in the HF + rosiglitazone group and lowest in the control group (Table 6). Despite the

high caloric intake of the HF + rosiglitazone group, it had the lowest feed efficiency among the

42

HF-fed groups. The HF + 1% mango had statistically similar feed efficiency to the HF +

rosiglitazone and HF groups (Table 6). The control group had the lowest feed efficiency hence,

the lowest body weight gain. Among the HF fed groups, HF + rosiglitazone had the lowest

weight gain and lowest final body weight followed by HF + 1% mango group. Mice in the HF +

mangiferin had the highest feed efficiency and final body weight which was statistically similar to

HF. In terms of tissue weights, rosiglitazone was able to minimize the expansion of white adipose

tissues and perirenal white adipose tissues due to high fat diet. Adipose tissue weight of mice in

the HF + rosiglitazone group was statistically similar to the control group (Table 6). Mice in the

HF + mangiferin group had the highest adipose tissue weights and were statistically similar to

HF. Mice in the HF+1% mango group had lower adipose tissue weights than the HF+mangiferin

group; however, it is also statistically similar to the HF group. There were no significant

differences in liver, spleen and kidney weights (Table 6).

Whole body composition

There were significant differences in body composition among the treatment groups

(Table 7). Mice receiving any of the HF diets have similar lean mass which was higher than the

control mice. Fat mass was also higher in the HF-fed groups compared to the control; however,

fat mass of the HF + rosiglitazone group was statistically similar to the control group. HF +

mangiferin had higher fat mass compared to HF+1% mango and HF + rosiglitazone groups

although statistically similar to the HF group. Mice in the HF+1% mango group had significantly

lower fat mass than the HF + mangiferin group but it was statistically similar to the HF group. All

HF-fed mice had higher total mass than the control. However, the HF + rosiglitazone and HF +

1% mango groups had significantly lower total mass than HF + mangiferin and HF groups.

Expressed as % of total mass, HF + rosiglitazone group had similar % body fat as the

control group. HF + mangiferin group had the highest % body fat and statistically similar to HF

43

group. HF + 1% mango group was statistically similar to HF group had approximately 10% lower

body fat in comparison to the HF group. The % lean mass for the control group was the highest

and it was statistically similar to the HF + rosiglitazone group. This was followed by HF + 1%

mango group and HF + mangiferin group had the lowest % lean mass (Table 7).

All HF-fed groups had lower bone mineral area and content compared to the control

group and there were no differences in any of the HF groups. There was no significant difference

in bone mineral density among the treatment groups (Table 7).

Clinical chemistry

Results of clinical chemistry parameters and liver and fecal lipids are shown in Table 8.

HF + mangiferin showed the highest plasma triglyceride concentration and statistically similar to

the HF group. Plasma triglycerides of HF + 1% mango and HF + rosiglitazone groups were

statistically similar to the control and HF groups. HF + 1% mango, HF + mangiferin, and HF

groups had similar plasma total cholesterol concentration. Only HF + rosiglitazone group had

similar plasma total cholesterol concentration to the control group. All the HF-fed groups had

higher HDL levels compared to control group, with HF + mangiferin group being the highest,

followed by HF, HF + 1% mango and HF + rosiglitazone groups. HF + rosiglitazone group had

the lowest level of non-esterified fatty acid (NEFA) and statistically different from all the other

treatment groups. Plasma NEFA in HF + 1% mango group was statistically similar to both the

control and HF groups while HF + mangiferin group had the highest plasma NEFA and also

statistically similar to the HF group.

There were no significant differences in fructosamine and glycated hemoglobin among

the treatment groups. However, there was significant difference in insulin among the treatment

groups. HF + mangiferin group had insulin level statistically similar to HF + 1% mango group.

44

Mice in the control, HF, HF + 1% mango, HF + rosiglitazone groups had statistically similar

plasma insulin concentration.

There was no significant difference in liver total lipid among the groups. However, fat

excretion was different with all HF groups having higher fecal total lipid than the control. The

control group had the lowest fecal total lipid and statistically different from all HF-fed groups.

Fecal total lipids of all the HF-fed mice were statistically the same.

Glucose tolerance test (GTT)

An intraperitoneal glucose tolerance test was performed after 30 and 60 days of dietary

treatments. Baseline blood glucose concentration after 30 days of treatment was similar for all

treatment groups (Figure 3a). Blood glucose concentrations were the same among the five

treatment groups five minutes post-glucose injection. Significant differences in blood glucose

were observed 15, 30 and 60 minutes post-injection. At 15 and 30 minutes, all HF fed groups had

similar blood glucose and were statistically higher than control group. However, there was

significant difference among the HF groups after 60 minutes of glucose injection. At this time

point, HF + mangiferin had the highest blood glucose and HF + rosiglitazone group was

statistically similar to the control group. HF + mango group had slightly lower blood glucose

concentration; statistically similar to HF + rosiglitazone group but was also not different from the

HF group. There were no significant differences in blood glucose concentrations among the

treatment groups two hours post-glucose injection. The total glucose area under curve after 30

days of dietary treatment was higher in all the HF-fed groups compared to the control and there

was no difference among the HF groups (Figure 3c).

Unlike the results of the 30 days glucose tolerance test, the glucose tolerance test after 60

days of dietary treatment showed significant differences among the treatment groups from

baseline to 120 minutes after the glucose injection (Figure 3b). The control group had the lowest

45

blood glucose concentration from baseline to 120 minutes. HF + rosiglitazone group consistently

reduced blood glucose and was statistically similar to the control group from baseline through

120 minutes. Mangiferin and mango were not effective in reducing blood glucose concentration

at all-time points post-glucose injection. The glucose AUC at 60 days of treatment were similar

for the control and HF + rosiglitazone groups and lower compared to the other HF groups. Unlike

rosiglitazone, mangiferin and mango was not able to improve glucose response in mice fed high

diet.

Histology

Figure 4 shows the results of liver and adipose tissue histology. HF group had larger

adipocytes compared to the control. HF +1% mango and HF+ mangiferin groups had slightly

smaller adipocytes compared to the HF group but not as small as HF + rosiglitazone group. HF +

rosiglitazone showed comparable adipocytes size to control group.

Liver histology shows diffuse cytoplasmic vacuolization, lipid droplets and steatosis in

the HF group compared to the control. Hepatocytes in the HF + 1% mango and HF+ rosiglitazone

groups appeared comparable to the control group, with no signs of steatosis or presence of lipid

droplets. HF + mangiferin group showed intermediate effects between the control group and HF

group as slight cytoplasmic vacuolization was observed.

46

Table 6: Effects of mango, rosiglitazone and mangiferin on food intake and body and tissue weights of mice fed high fed diets for two months.

Parameters AIN-93 High Fat High Fat + 1%

Mango

High Fat +

Mangiferin

High Fat +

Rosiglitazone

P value

Body weight (g)

Initial 21.17±1.92 21.18±1.85 21.25±1.88 21.29±1.46 21.69±1.09 0.9810

Final 25.85±2.52d 35.47±3.70

ab 32.24±4.02

bc 36.24±3.64

a 30.83±2.20

c <.0001

Weight gain (g) 4.68±2.66d 14.29±3.62

ab 10.99±4.31

bc 14.95±4.08

a 9.14±1.82

c <.0001

Caloric intake

(kcal/d)

11.36±0.75c 14.77±0.69

b 14.51±1.08

b 14.40±0.92

b 15.85±1.18

a <.0001

Food intake

(g/d)

3.09±0.20a 2.64±0.12

c 2.65±0.20

c 2.58±0.16

c 2.84±0.21

b <.0001

Feed efficiency

(g/g diet )

1.51±0.86d 5.41±1.37

ab 4.14±1.62

bc 5.80±1.58

a 3.22±0.64

c <.0001

Feed efficiency

(g/kal diet )

0.41±0.23d 1.26±0.32

ab 0.97±0.38

bc 1.32±0.36

a 0.80±0.16c <.0001

Tissue weights (mg)

White adipose

tissue

461.4±143.6c

1631.2±521.4ab

1275.7±622.2b 2000.0±486.7

a 515.0±251.1

c <.0001

Perirenal white

adipose tissue

113.3±80.8b 676.0±313.8

a 557.5±339.8

ab 872.5±342.3

a 140.0±62.5

b 0.012

Liver 911.4±169.9 1095.0±264.5 1057.1±85.0 1061.3±130.4 963.3±137.9 0.2475

Spleen 108.6±56.7 87.5±19.8 125.0±75.0 81.3±18.9 105.0±22.6 0.3520

Kidneys 310.0±43.4 350.0±36.6 318.0±28.6 332.5±29.2 320.0±23.7 0.1948

Values are mean ± SD, n=8/group; within a row, values that do not share the same letters are significantly (P<0.05) different from each other.

47

Table 7: Effects of mango, rosiglitazone and mangiferin on whole body composition, bone mineral area

(BMA), content (BMC) and density (BMD) of mice fed high fat diet for two months.

Parameters AIN-93M High Fat High Fat +

1% Mango

High Fat +

Mangiferin

High Fat +

Rosiglitazone

P value

Lean mass

(g)

17.4±1.7b 22.0±1.5

a 20.5±1.8

a 21.6±1.6

a 21.0±0.7

a <0.0001

Fat mass(g) 5.3±1.1c 11.5±2.4

ab 9.5±2.8

b 12.8±2.3

a 6.6±1.7

c <0.0001

Total

mass(g)

22.7±2.5c 33.5±3.6

a 30.0±3.9

b 34.4±3.5

a 27.5±2.2

b <0.0001

% fat 23.2±3.2c 34.2±3.9

ab 31.0±6.4

b 37.0±3.4

a 23.7±4.1

c <0.0001

% lean 76.8±3.2a 65.8±4.0

bc 69.0±6.5

b 63.0±3.4

c 76.4±4.0

a <0.0001

BMA(cm2) 11.5±0.5

a 10.1±0.5

b 10.4±0.9

b 9.7±0.6

b 10.3±0.8

b 0.0001

BMC (mg) 583.4±57.4a 505.4±26.4

b 516.6±58.9

b 481.6±52.3

b 493.8±414.7

b 0.0025

BMD

(mg/cm2)

50.8±3.3 50.3±1.0 49.8±2.0

49.6±2.3 47.7±1.3 0.1219

Values are mean ± SD, n=8/group; within a row, values that do not share the same letters are

significantly (P<0.05) different from each other

48

Table 8: Effects of mango, rosiglitazone and mangiferin on clinical chemistry parameters, and liver and

fecal total lipid of mice fed high fat diet for two months

Values are mean ± SD, n=8/group; within a row, values that do not share the same letters are significantly

(P<0.05) different from each other. NEFA= non-esterified fatty acid; HbA1c = glycated hemoglobin;

HDL= high density lipoprotein.

Parameters AIN-93M High Fat High Fat +

1% Mango

High Fat +

Mangiferin

High Fat +

Rosiglitazone

P value

Plasma

Triglyceride

(mg/dL)

22.4±2.1b 29.9±8.7

ab 25.7±9.9

b 36.7±9.8

a 22.8±3.6

b 0.0051

Total cholesterol

(mg/dL)

83.6±26.1c 110.2±24.6

ab 118.7±36.4

a 116.4±18.3

a 83.8±5.5

bc 0.0154

HDL-cholesterol

(mg/dL)

31.9±12.9c 43.1±7.0

ab 41.6±12.6

ab 46.3±4.9

a 35.3±2.1

bc 0.0254

NEFA(mEq/L) 0.65±0.12b 0.74±0.08

ab 0.64±0.15

b 0.80±0.13

a 0.42±0.06

c <.0001

Fructosamine

(µmol/L)

226.0±68.3 215.3±30.5 182.0±46.2 228.3±37.0 244.5±53.4 0.2119

Glucose

(mg/dL)

189±29bc

220±44ab

236±32a 215±37

ab 149±48

c 0.0028

HbA1c (%) 2.97±0.70 3.44±0.60 3.46±0.39 3.23±1.25 3.98±0.38 0.1849

Insulin (ng/mL) 0.27±0.03b 0.34±0.05

b 0.34±0.09

ab 0.50±0.18

a 0.24±0.05

b 0.0348

Total Lipid ( mg/g)

Liver 106.08±

77.08

94.63±

58.36

171.56±

54.33

135.63±

44.38

119.98±

60.39

0.4941

Fecal 18.73±

3.13b

49.43±

2.29a

54.12±

4.57a

49.86±

11.01a

52.82±

10.62a

<0.0001

49

Figure 2 : Effects of mango, rosiglitazone and mangiferin on weekly body weights of mice fed with

high fed diets for two months.

(*) Indicates significantly (P<0.05) different from each other. Weekly body weights of the control

group (AIN-93) are statistically different from all the HF groups from week 1 through week 8. Weekly

body weights of the mangiferin group are statistically similar to the HF group throughout the entire

treatment period. The 1% mango diet has an intermediate effect on weekly body weights, being similar

to the rosiglitazone and HF groups from week 1 through week 8.

50

Figure 3: Effects of mango, rosiglitazone and mangiferin on glucose tolerance (a) after 30 days of

treatment (b) after 60 days of treatment and (c) total glucose area under the curve

(*) Indicates significantly (P<0.05) different from each other.

(mg/dL)

(min)

51

Bars are mean ±SD, n=8/group; bars that do not share the same letters are significantly (P<0.05) different

from each other. All comparison among groups at 30 d uses small letters and 60 d comparison are shown

with capital letters.

52

Figure 4: Effects of mango, rosiglitazone and mangiferin on liver and adipose tissue histology of

mice fed with high fed diets for two months.

Dietary

treatment group

Liver histology White adipose tissue histology

AIN-93

High Fat

HF+ 1% Mango

HF + Mangiferin

HF+

Rosiglitazone

53

CHAPTER V

DISCUSSION

The leaves and stem bark extracts of mango have been well-documented for its anti-

diabetic [26, 191-195] and hypolipidemic [26, 29, 196] properties. As mangiferin is the major

polyphenolic constituent in the mango leaves and stem bark, this bioactive compound has become

the focus for many researchers. To our knowledge, no studies have compared the effect of mango

pulp to mangiferin and rosiglitazone in improving clinical parameters associated with metabolic

syndrome. How much mangiferin contributes to the positive health effect of mango pulp is still

unknown. We have previously reported blood glucose lowering properties and body fat

modulation by 1% freeze-dried mango pulp in mice fed high fat diet [197]. However, whether

mangiferin is a major contributor to these positive effects of mango pulp warrants further

investigation. Hence, the present study compared the effects of mangiferin and freeze-dried

mango pulp on body composition, blood glucose and plasma lipid in mice fed high fat diet.

Additionally, the effects of freeze-dried mango and mangiferin were compared to rosiglitazone on

mitigating MetS risk factors.

In this study, all HF-fed groups had higher body weights compared to the control group

due to higher caloric intake and feed efficiency, which was consistent with previous studies on

mice [198-201]. Increased feed efficiency has been shown to associate with adipocyte hyperplasia

[200], which was consistent with the findings of this study . Mangiferin had higher feed

54

efficiency than mango group and rosiglitazone. Hence, mangiferin had highest adipose tissue

weights, number of expanded adipocytes and percentage of fat compared to 1% mango and

rosiglitazone groups. Therefore, mangiferin combined with high fat diet (60% calories from fat)

seemed to not confer any benefit in preventing fat accumulation. Lucas and colleagues [197]

showed that 1% freeze-dried mango pulp modulated body composition in mice fed high fat diet.

In contrast, a slight reduction in % body fat (approximately 10%) was observed in the present

study with the same dose of freeze-dried mango; however, the % body fat was not statistically

different from the HF group. The feed efficiency for the mango group was statistically similar to

the rosiglitazone group but was also similar to the HF group. This data shows that mango pulp

was more effective than mangiferin in modulating body composition. This indicates that other

components of mango, aside from mangiferin, maybe responsible for the positive effect of mango

pulp on body composition.

This study also compared the effects of mangiferin and mango to rosiglitazone on

glucose homeostasis. Earlier findings by Ichiki et.al [27] showed mangiferin (30 mg/kg and 90

mg/kg) decreased blood glucose in KKAy mice, an animal model of non-insulin dependent

diabetes mellitus (NIDDM). They also found that the glucose-lowering property of mangiferin at

90 mg/kg was more pronounced when compared to tolbutamide, a synthetic antidiabetic drug.

Unlike the findings of Ichiki, our findings do not support glucose lowering property of

mangiferin. Mangiferin was not able to lower fasting blood glucose or improve glucose tolerance

in mice fed high fat diet. The blood glucose and glucose AUC of mice in the mangiferin group

was statistically similar to mango and HF and significantly higher compared to rosiglitazone. In

addition to mangiferin, we also did not observe a glucose-modulating property of mango pulp.

This finding is not in agreement with the findings of Lucas et.al [197] that demonstrated 1%

freeze-dried mango pulp was able to reduce blood glucose during glucose tolerance test in mice

fed high fat diet. They also observed that mice that received the 1% freeze-dried mango pulp had

55

lower blood glucose lowering property than those that received the rosiglitazone treatment. This

discrepancy might be due to the instability of the active component in mango that possesses

glucose lowering property. The mango pulp used in the present study has been frozen for almost a

year compared to a fresh batch used in the study by Lucas et. al..

In this study, we also compared the effects of mangiferin, mango and rosiglitazone on

lipid profile. Previous studies [26, 29, 172] have shown that mangiferin was able to lower plasma

triglycerides in chemically-induced diabetes or diet- induced hyperlipidemia in rodents. However,

our study found that mangiferin was not able to modulate plasma triglycerides as it had the

highest plasma trglyceride concentrations among the HF-fed mice and was statistically similar to

the HF group. Although mangiferin was not able to modulate plasma triglycerides, mice in the

mango group had slightly lower plasma triglycerides. They had plasma triglyceride

concentrations similar to rosiglitazone albeit not statistically different from the HF group.

Muruganandan and colleagues [26] and Miura et.al [172] also found that mangiferin decreased

plasma total cholesterol in streptozotocin-induced diabetic rats and animal models of type 2

diabetes mellitus. Inconsistent with their findings, we found that both mangiferin and mango had

no hypocholesterolemic effect.

The study by Guo et.al [29] was very similar to our methodology as they studied effects

of mangiferin on lipid profiles in hamsters fed high fat diet. The hypolipidemic property of

mangiferin in this animal model might be attributed to the mangiferin doses that was used (50

mg/kg and 150 mg/kg body weight) compared to 0.003 mg/kg body weight in our study. This

mangiferin dose was equivalent to the mangiferin content in the 1% mango pulp and it was

derived using food intake divided by final body weight in the mangiferin group and times with a

dose of 0.044 mg/kg diet. Additionally, Miura et.al [172] also demonstrated that mangiferin (30

mg/kg body weight) was able to decrease blood cholesterol and triglyceride levels in KK-Ay

56

mice. Therefore, pharmacological doses of mangiferin might be necessary to produce these

positive effects on lipid profile and other MetS risk factors.

Another possible explanation for the lack of effect of mangiferin on the assessed clinical

parameters may be due to low biological activity or antioxidative capacity of isolated phenolic

compounds compared to the synergistic effects of the combination of the polyphenolic

compounds found in mango pulp [149, 158, 202]. Bohm and colleagues [203] examined the

antioxidant capacity of the individual and combination of ß-carotene, vitamin E and vitamin C on

the integrity of human lymphoid cells when the cells were excited with pulse radiolysis to

generate nitrogen dioxide free radicals. They observed that the combination treatment of ß-

carotene, vitamin E and vitamin C showed less membrane destruction compared to individual

components, suggesting synergistic interaction among components is crucial for exhibiting

antioxidant properties. In our study, the body composition, glucose and lipid profile in mangiferin

consistently showed similar effects to HF, further suggesting the importance of the synergistic

properties in mitigating MetS risk factors.

Considering that the present study used similar methodology as Lucas et.al [197], the

weak effect of the 1% mango in blood glucose and lipid modulation in the present study suggest

that factors other than methodology could explain the different outcomes. One explanation is the

stability of the active compounds in the mango powder. In this study, the mango used was a

combination of different batches of freeze-dried mango powder from previous years. This might

have attributed to the decreased activity of the antioxidant compounds in mango, leading to

insignificant findings. Antioxidant compounds like ascorbic acid, β-carotene and other phenolic

compounds found in mango have been well-documented to exhibit health-promoting properties

including anti-inflammatory and antiatherosclerosis in improving the risk factors of developing

metabolic syndrome [146, 188]. Longer storage may have reduced the activity of these

antioxidant compounds.

57

Hymavathi and Khader [163] studied the effects of storage duration and packaging

materials on the changes in phenolic compounds and other bioactive compounds in mango

powder. They found positive correlation between the degradation of total carotene and ascorbic

acid with storage duration. Furthermore, dehydrated products like freeze-dried mango powder

were more susceptible to deterioration or oxidation due to increased porosity and surface area

[204]. Additionally, the stability of bioactive compound such as ascorbic acid in freeze-dried

mango powder was negatively correlated with moisture content and oxygen [205]. Packaging

materials that are completely impermeable to the air or water are crucial in minimizing the loss of

antioxidant compounds in the mango powder. [205].

Although the mango powder from the present study and that of Lucas et.al was from the

same Tommy Atkins cultivar, the carotenoid contents may have differed due to different harvest

locations [146, 161]; this factor could not be neglected considering mango powder in the present

study was from combination of different batches of mango powder. It has been shown that hot

and moderate climate affected the carotenoid content in the mango [146]. Godoy and Rodriguez-

Amaya [206] demonstrated that Tommy Atkins mango grown in hot areas like Mato Grosso,

Brazil had more β-carotene content compared to moderate areas like Sao Paulo, Brazil.

Furthermore, a diverse range of amounts of gallotannin in Tommy Atkins mango pulp was

reported even though the mangos were harvested from subtropical countries [188]. The

inconsistencies in the quantity of phenolic compounds in mangos occurred although they were

from the same cultivar, leading to inevitable variability in different batches of mango.

The ripeness of the mango is also critical in interpreting our results. It has been

documented that the ripening stage of mango caused prominent quantitative changes of total

carotenoid content [146]. Gil et.al [170] examined the compositional changes in the ripening

stage of mango pulp using nuclear magnetic resonance spectroscopy (NMRS). The researchers

observed that shikimic acid was the major constituent in unripe mangos, and gradually

58

diminished with ripening. During the early ripening stage, small metabolites like niacin and

phenylalanine were shown and when mango reached final ripening stage, formation of

polyphenolic compounds was detected.

In summary, mangiferin had lesser beneficial effect than mango but both mangiferin and

mango showed less beneficial effects in modulating body composition, triglyceride levels and

total cholesterol when compared to rosiglitazone. Rosiglitazone is known to increase insulin

sensitivity by up-regulating adiponectin secretion by adipocytes [105], leading to increased FA

oxidation and declined influx of FFA in liver and muscle cells [108-110]. Therefore, less fat

deposition reduced the adipose tissue weights and hyperplasia, leading to body composition

modulation in mice with rosiglitazone treatment.

Our findings also suggest that mangiferin (0.003 mg/kg) is not a key contributor to the

beneficial effects of 1% mango. Mangiferin, an isolated phenolic compound, showed weaker

bioactivity compared to the synergistic interactions among all phenolic or antioxidant compounds

found in mango pulp. Our data further demonstrated the importance of the interactions among

antioxidant and polyphenolic compounds in exhibiting health- promoting properties.

The main limitation of this study as discussed above is the use of a mixture of different

batches of freeze-dried mango which can come from different harvest locations and the different

stages of fruit maturity. Moreover, extended storage and mango packaging may have contributed

to changes in the bioactive components of mango, leading to decreased antioxidant potential in

ameliorating MS risk factors. Another limitation is the qualitative assessment of the liver and

adipose tissue histology. The quantification of cytoplasmic vacuolization, lipid droplets and

steatosis in liver histology was not assessed in this study. Moreover, the quantitation of adipose

tissue hypertrophy and hyperplasia was not performed. Hence, interpretation of this data is

subject to investigator bias.

59

In conclusion, we reject the null hypothesis that there is no difference between

mangiferin, mango and rosiglitazone in (1) modulating body composition; (2) lowering glucose

concentration and glucose homeostasis, (3) modulating other clinical parameters in mice fed high

fat diet.

60

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VITA

CHEEYEN LAU

Candidate for the Degree of

Master of Science

Thesis: EFFECTS OF MANGIFERIN IN IMPROVING METABOLIC SYNDROME

RISK FACTORS IN MICE FED HIGH FAT DIET

Major Field: Nutritional Science

Biographical:

Education:

Completed the requirements for the Master of Science in nutritional science at

Oklahoma State University, Stillwater, Oklahoma in May, 2012.

Completed the requirements for the Bachelor of Science in Medical Bioscience

at Monash University, Sunway City, Malaysia in 2009.

Experience:

Graduate assistant, Oklahoma State University, Division of Engineering

Technology, 2010-2012.

Graduate research assistant, Oklahoma State University, Nutritional Sciences

Department, 2010-2012.

ADVISER’S APPROVAL: Dr. Edralin A. Lucas

Name: CHEEYEN LAU Date of Degree: May, 2012

Institution: Oklahoma State University Location: Stillwater, Oklahoma

Title of Study: EFFECTS OF MANGIFERIN IN IMPROVING METABOLIC

SYNDROME RISK FACTORS IN MICE FED HIGH FAT DIET

Pages in Study: 72 Candidate for the Degree of Master of Science

Major Field: Nutritional Science

Scope and Method of Study: This study compared the effects of freeze-dried mango pulp,

mangiferin, and rosiglitazone (a glucose-lowering drug) on ameliorating risk

factors associated with metabolic syndrome (MetS) in mice fed high fat (HF) diet.

Two-month old male C57BL/6 mice were randomly assigned to one of the dietary

treatment groups (n=8/group) for 60 days: AIN-control (10 % fat calories), HF

(60 % fat calories), HF+1 % (w/w) freeze dried mango, HF + mangiferin (0.044

mg/kg diet), and HF + rosiglitazone (50 mg/kg diet). Whole body composition,

glucose tolerance test (GTT), clinical chemistry, and liver and adipose tissue

histology were assessed.

Findings and Conclusions: Mangiferin did not modulate body composition, glucose and

lipid parameters compared to mango and rosiglitazone in mice fed HF diet.

Mango pulp showed more positive effects compared to mangiferin in modulating

body composition and blood lipids. Our findings suggest that mangiferin is not a

key contributor to the beneficial effects of mango pulp in modulating MetS risk

factors. Furthermore, our findings demonstrate the importance of synergistic

interactions of the bioactive compounds in mango pulp.